Ferredoxin reduction by hydrogen with iron functions as an evolutionary precursor of flavin-based electron bifurcation.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin-the physiological donor of electrons in the acetyl-CoA pathway-with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? Here, we show that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pasteurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth's crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

Purification and structural characterization of the Na+-translocating ferredoxin: NAD+ reductase (Rnf) complex of Clostridium tetanomorphum.

Various microbial metabolisms use H+/Na+-translocating ferredoxin:NAD+ reductase (Rnf) either to exergonically oxidize reduced ferredoxin by NAD+ for generating a transmembrane electrochemical potential or reversely to exploit the latter for producing reduced ferredoxin. For cryo-EM structural analysis, we elaborated a quick four-step purification protocol for the Rnf complex from Clostridium tetanomorphum and integrated the homogeneous and active enzyme into a nanodisc. The obtained 4.27 Å density map largely allows chain tracing and redox cofactor identification complemented by biochemical data from entire Rnf and single subunits RnfB, RnfC and RnfG. On this basis, we postulated an electron transfer route between ferredoxin and NAD via eight [4Fe-4S] clusters, one Fe ion and four flavins crossing the cell membrane twice related to the pathway of NADH:ubiquinone reductase. Redox-coupled Na+ translocation is provided by orchestrating Na+ uptake/release, electrostatic effects of the assumed membrane-integrated FMN semiquinone anion and accompanied polypeptide rearrangements mediated by different redox steps.

Energy Conservation in Fermentations of Anaerobic Bacteria.

Anaerobic bacteria ferment carbohydrates and amino acids to obtain energy for growth. Due to the absence of oxygen and other inorganic electron acceptors, the substrate of a fermentation has to serve as electron donor as well as acceptor, which results in low free energies as compared to that of aerobic oxidations. Until about 10 years ago, anaerobes were thought to exclusively use substrate level phosphorylation (SLP), by which only part of the available energy could be conserved. Therefore, anaerobes were regarded as unproductive and inefficient energy conservers. The discovery of electrochemical Na+ gradients generated by biotin-dependent decarboxylations or by reduction of NAD+ with ferredoxin changed this view. Reduced ferredoxin is provided by oxidative decarboxylation of 2-oxoacids and the recently discovered flavin based electron bifurcation (FBEB). In this review, the two different fermentation pathways of glutamate to ammonia, CO2, acetate, butyrate and H2 via 3-methylaspartate or via 2-hydroxyglutarate by members of the Firmicutes are discussed as prototypical examples in which all processes characteristic for fermentations occur. Though the fermentations proceed on two entirely different pathways, the maximum theoretical amount of ATP is conserved in each pathway. The occurrence of the 3-methylaspartate pathway in clostridia from soil and the 2-hydroxyglutarate pathway in the human microbiome of the large intestine is traced back to the oxygen-sensitivity of the radical enzymes. The coenzyme B12-dependent glutamate mutase in the 3-methylaspartate pathway tolerates oxygen, whereas 2-hydroxyglutaryl-CoA dehydratase is extremely oxygen-sensitive and can only survive in the gut, where the combustion of butyrate produced by the microbiome consumes the oxygen and provides a strict anaerobic environment. Examples of coenzyme B12-dependent eliminases are given, which in the gut are replaced by simpler extremely oxygen sensitive glycyl radical enzymes.

Spectral deconvolution of redox species in the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from Megasphaera elsdenii. A flavin-dependent bifurcating enzyme.

We have undertaken a spectral deconvolution of the three FADs of EtfAB/bcd to the spectral changes seen in the course of reduction, including the spectrally distinct anionic and neutral semiquinone states of electron-transferring and bcd flavins. We also demonstrate that, unlike similar systems, no charge-transfer complex is observed on titration of the reduced M. elsdenii EtfAB with NAD+. Finally, and significantly, we find that removal of the et FAD from EtfAB results in an uncrossing of the half-potentials of the bifurcating FAD that remains in the protein, as reflected in the accumulation of substantial FAD•- in the course of reductive titrations of the depleted EtfAB with sodium dithionite.

Flavins in the electron bifurcation process.

The discovery of a new energy-coupling mechanism termed flavin-based electron bifurcation (FBEB) in 2008 revealed a novel field of application for flavins in biology. The key component is the bifurcating flavin endowed with strongly inverted one-electron reduction potentials (FAD/FAD•- ≪ FAD•-/FADH-) that cooperatively transfers in its reduced state one low and one high-energy electron into different directions and thereby drives an endergonic with an exergonic reduction reaction. As energy splitting at the bifurcating flavin apparently implicates one-electron chemistry, the FBEB machinery has to incorporate prior to and behind the central bifurcating flavin 2e-to-1e and 1e-to-2e switches, frequently also flavins, for oxidizing variable medium-potential two-electron donating substrates and for reducing high-potential two-electron accepting substrates. The one-electron carriers ferredoxin or flavodoxin serve as low-potential (high-energy) electron acceptors, which power endergonic processes almost exclusively in obligate anaerobic microorganisms to increase the efficiency of their energy metabolism. In this review, we outline the global organization of FBEB enzymes, the functions of the flavins therein and the surrounding of the isoalloxazine rings by which their reduction potentials are specifically adjusted in a finely tuned energy landscape.

Modulations of the reduction potentials of flavin-based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow.

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, ß-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces ß-FAD to ß-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of ß-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from ß-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.

Rapid kinetics reveal surprising flavin chemistry in bifurcating electron transfer flavoprotein from Acidaminococcus fermentans.

Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. ß-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low-potential ferredoxin and the other to the high-potential α-FAD semiquinone (α-FAD•-). The resultant α-FAD hydroquinone (α-FADH-) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD•- to monitor formation of α-FAD•- or α-FADH-, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to ß-FAD at a rate of 920 s-1, yielding the charge-transfer complex NAD+:ß-FADH- with an absorbance maximum at 650 nm. ß-FADH- bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD•-. With α-FAD•-, the reduction of ß-FAD with NADH was 1500 times slower. Reduction of ß-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD•-. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.

Molecular and Low-Resolution Structural Characterization of the Na+-Translocating Glutaconyl-CoA Decarboxylase From Clostridium symbiosum.

Some anaerobic bacteria use biotin-dependent Na+-translocating decarboxylases (Bdc) of ß-keto acids or their thioester analogs as key enzymes in their energy metabolism. Glutaconyl-CoA decarboxylase (Gcd), a member of this protein family, drives the endergonic translocation of Na+ across the membrane with the exergonic decarboxylation of glutaconyl-CoA (ΔG 0' ≈-30 kJ/mol) to crotonyl-CoA. Here, we report on the molecular characterization of Gcd from Clostridium symbiosum based on native PAGE, size exclusion chromatography (SEC) and laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). The obtained molecular mass of ca. 400 kDa fits to the DNA sequence-derived mass of 379 kDa with a subunit composition of 4 GcdA (65 kDa), 2 GcdB (35 kDa), GcdC1 (15 kDa), GcdC2 (14 kDa), and 2 GcdD (10 kDa). Low-resolution structural information was achieved from preliminary electron microscopic (EM) measurements, which resulted in a 3D reconstruction model based on negative-stained particles. The Gcd structure is built up of a membrane-spanning base primarily composed of the GcdB dimer and a solvent-exposed head with the GcdA tetramer as major component. Both globular parts are bridged by a linker presumably built up of segments of GcdC1, GcdC2 and the 2 GcdDs. The structure of the highly mobile Gcd complex represents a template for the global architecture of the Bdc family.

Physiological limits to life in anoxic subseafloor sediment.

In subseafloor sediment, microbial cell densities exponentially decrease with depth into the fermentation zone. Here, we address the classical question of 'why are cells dying faster than they are growing?' from the standpoint of physiology. The stoichiometries of fermentative ATP production and consumption in the fermentation zone place bounds on the conversion of old cell biomass into new. Most fermentable organic matter in deep subseafloor sediment is amino acids from dead cells because cells are mostly protein by weight. Conversion of carbon from fermented dead cell protein into methanogen protein via hydrogenotrophic and acetoclastic methanogenesis occurs at ratios of ∼200:1 and 100:1, respectively, while fermenters can reach conversion ratios approaching 6:1. Amino acid fermentations become thermodynamically more efficient at lower substrate and product concentrations, but the conversion of carbon from dead cell protein into fermenter protein is low because of the high energetic cost of translation. Low carbon conversion factors within subseafloor anaerobic feeding chains account for exponential declines in cellular biomass in the fermentation zone of anoxic sediments. Our analysis points to the existence of a life-death transition zone in which the last biologically catalyzed life processes are replaced with purely chemical reactions no longer coupled to life.

Structural and Functional Characterization of an Electron Transfer Flavoprotein Involved in Toluene Degradation in Strictly Anaerobic Bacteria.

(R)-Benzylsuccinate is the characteristic initial intermediate of anaerobic toluene metabolism, which is formed by a radical-type addition of toluene to fumarate. Its further degradation proceeds by activation to the coenzyme A (CoA)-thioester and ß-oxidation involving a specific (R)-2-benzylsuccinyl-CoA dehydrogenase (BbsG) affiliated with the family of acyl-CoA dehydrogenases. In this report, we present the biochemical properties of electron transfer flavoproteins (ETFs) from the strictly anaerobic toluene-degrading species Geobacter metallireducens and Desulfobacula toluolica and the facultatively anaerobic bacterium Aromatoleum aromaticum We determined the X-ray structure of the ETF paralogue involved in toluene metabolism of G. metallireducens, revealing strong overall similarities to previously characterized ETF variants but significantly different structural properties in the hinge regions mediating conformational changes. We also show that all strictly anaerobic toluene degraders utilize one of multiple genome-encoded related ETF paralogues, which constitute a distinct clade of similar sequences in the ETF family, for ß-oxidation of benzylsuccinate. In contrast, facultatively anaerobic toluene degraders contain only one ETF species, which is utilized in all ß-oxidation pathways. Our phylogenetic analysis of the known sequences of the ETF family suggests that at least 36 different clades can be differentiated, which are defined either by the taxonomic group of the respective host species (e.g., clade P for Proteobacteria) or by functional specialization (e.g., clade T for anaerobic toluene degradation).IMPORTANCE This study documents the involvement of ETF in anaerobic toluene metabolism as the physiological electron acceptor for benzylsuccinyl-CoA dehydrogenase. While toluene-degrading denitrifying proteobacteria use a common ETF species, which is also used for other ß-oxidation pathways, obligately anaerobic sulfate- or ferric-iron-reducing bacteria use specialized ETF paralogues for toluene degradation. Based on the structure and sequence conservation of these ETFs, they form a new clade that is only remotely related to the previously characterized members of the ETF family. An exhaustive analysis of the available sequences indicated that the protein family consists of several closely related clades of proven or potential electron-bifurcating ETF species and many deeply branching nonbifurcating clades, which either follow the host phylogeny or are affiliated according to functional criteria.

Formation of 3-hydroxyglutaric acid in glutaric aciduria type I: in vitro participation of medium chain acyl-CoA dehydrogenase.

3-Hydroxyglutaric acid (3-OH-GA) in urine has been identified as the most reliable diagnostic marker for glutaric aciduria type I (GA I). We showed that hydratation of glutaconyl-CoA to 3-hydroxyglutaryl-CoA, which is subsequently hydrolyzed to 3-OH-GA, is efficiently catalyzed by 3-methylglutaconyl-CoA hydratase (3-MGH). We have now investigated whether mitochondrial acyl-CoA-dehydrogenases can convert glutaryl-CoA to glutaconyl-CoA. Short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) accepted glutaryl-CoA as a substrate. The highest k cat of glutaryl-CoA was found for MCAD (0.12 ± 0.01 second-1) and was about 26-fold and 52-fold higher than those of LCAD and SCAD, respectively. The turnover of MCAD for glutaryl-CoA was about 1.5% of that of its natural substrate octanoyl-CoA. Despite high K m (above 600 µM) and low turnover rate, the oxidation of glutaryl-CoA by MCAD in combination with 3-MGH could explain the urinary concentration of 3-OH-GA in GA I patients.

Enzymatic Reactions Involving Ketyls: From a Chemical Curiosity to a General Biochemical Mechanism.

Ketyls are radical anions with nucleophilic properties. Ketyls obtained by enzymatic one-electron reduction of thioesters were proposed as intermediates for the dehydration of (R)-2-hydroxyacyl-CoA to (E)-2-enoyl-CoA. This concept was extended to the Birch-like reduction of benzoyl-CoA to 1,5-cyclohexadienecarboxyl-CoA. Nature uses two methods to achieve the therefore required low reduction potentials of less than -600 mV, either by an ATP-driven electron transfer similar to that catalyzed by the iron protein of nitrogenase or by electron bifurcation. Ketyls formed by thiyl radical-initiated oxidation of alcohols followed by deprotonation are involved in coenzyme B12-independent diol dehydratases, other glycyl radical enzymes mediating key reactions in the degradations of choline, taurine, and 4-hydroxyproline, and all three classes of ribonucleotide reductases. A special case is the dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which most likely proceeds via an oxidation to an allylic ketyl but requires neither a strong reductant nor an external radical generator.

Flavin-Based Electron Bifurcation, Ferredoxin, Flavodoxin, and Anaerobic Respiration With Protons (Ech) or NAD+ (Rnf) as Electron Acceptors: A Historical Review.

Flavin-based electron bifurcation is a newly discovered mechanism, by which a hydride electron pair from NAD(P)H, coenzyme F420H2, H2, or formate is split by flavoproteins into one-electron with a more negative reduction potential and one with a more positive reduction potential than that of the electron pair. Via this mechanism microorganisms generate low- potential electrons for the reduction of ferredoxins (Fd) and flavodoxins (Fld). The first example was described in 2008 when it was found that the butyryl-CoA dehydrogenase-electron-transferring flavoprotein complex (Bcd-EtfAB) of Clostridium kluyveri couples the endergonic reduction of ferredoxin (E0' = -420 mV) with NADH (-320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (-10 mV) with NADH. The discovery was followed by the finding of an electron-bifurcating Fd- and NAD-dependent [FeFe]-hydrogenase (HydABC) in Thermotoga maritima (2009), Fd-dependent transhydrogenase (NfnAB) in various bacteria and archaea (2010), Fd- and H2-dependent heterodisulfide reductase (MvhADG-HdrABC) in methanogenic archaea (2011), Fd- and NADH-dependent caffeyl-CoA reductase (CarCDE) in Acetobacterium woodii (2013), Fd- and NAD-dependent formate dehydrogenase (HylABC-FdhF2) in Clostridium acidi-urici (2013), Fd- and NADP-dependent [FeFe]-hydrogenase (HytA-E) in Clostridium autoethanogrenum (2013), Fd(?)- and NADH-dependent methylene-tetrahydrofolate reductase (MetFV-HdrABC-MvhD) in Moorella thermoacetica (2014), Fd- and NAD-dependent lactate dehydrogenase (LctBCD) in A. woodii (2015), Fd- and F420H2-dependent heterodisulfide reductase (HdrA2B2C2) in Methanosarcina acetivorans (2017), and Fd- and NADH-dependent ubiquinol reductase (FixABCX) in Azotobacter vinelandii (2017). The electron-bifurcating flavoprotein complexes known to date fall into four groups that have evolved independently, namely those containing EtfAB (CarED, LctCB, FixBA) with bound FAD, a NuoF homolog (HydB, HytB, or HylB) harboring FMN, NfnB with bound FAD, or HdrA harboring FAD. All these flavoproteins are cytoplasmic except for the membrane-associated protein FixABCX. The organisms-in which they have been found-are strictly anaerobic microorganisms except for the aerobe A. vinelandii. The electron-bifurcating complexes are involved in a variety of processes such as butyric acid fermentation, methanogenesis, acetogenesis, anaerobic lactate oxidation, dissimilatory sulfate reduction, anaerobic- dearomatization, nitrogen fixation, and CO2 fixation. They contribute to energy conservation via the energy-converting ferredoxin: NAD+ reductase complex Rnf or the energy-converting ferredoxin-dependent hydrogenase complex Ech. This Review describes how this mechanism was discovered.

Flavin-Based Electron Bifurcation, A New Mechanism of Biological Energy Coupling.

There are two types of electron bifurcation (EB), either quinone- or flavin-based (QBEB/FBEB), that involve reduction of a quinone or flavin by a two-electron transfer and two reoxidations by a high- and low-potential one-electron acceptor with a reactive semiquinone intermediate. In QBEB, the reduced low-potential acceptor (cytochrome b) is exclusively used to generate ΔµH+. In FBEB, the "energy-rich" low-potential reduced ferredoxin or flavodoxin has dual function. It can give rise to ΔµH+/Na+ via a ferredoxin:NAD reductase (Rnf) or ferredoxin:proton reductase (Ech) or conducts difficult reductions such as CO2 to CO. The QBEB membrane complexes are similar in structure and function and occur in all domains of life. In contrast, FBEB complexes are soluble and occur only in strictly anaerobic bacteria and archaea (FixABCX being an exception). The FBEB complexes constitute a group consisting of four unrelated families that contain (1) electron-transferring flavoproteins (EtfAB), (2) NAD(P)H dehydrogenase (NuoF homologues), (3) heterodisulfide reductase (HdrABC) or HdrABC homologues, and (4) NADH-dependent ferredoxin:NADP reductase (NfnAB). The crystal structures and electron transport of EtfAB-butyryl-CoA dehydrogenase and NfnAB are compared with those of complex III of the respiratory chain (cytochrome bc1), whereby unexpected common features have become apparent.

The semiquinone swing in the bifurcating electron transferring flavoprotein/butyryl-CoA dehydrogenase complex from Clostridium difficile.

The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and ß-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces ß-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH-, which reduces crotonyl-CoA.

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

Complete Genome Sequence of the Amino Acid-Fermenting Clostridium propionicum X2 (DSM 1682).

Clostridium propionicumis a strict anaerobic, Gram positive, rod-shaped bacterium that belongs to the clostridial cluster XIVb. The genome consists of one replicon (3.1 Mb) and harbors 2,936 predicted protein-encoding genes. The genome encodes all enzymes required for fermentation of the amino acids α-alanine, ß-alanine, serine, threonine, and methionine.

Reduction of Flavodoxin by Electron Bifurcation and Sodium Ion-dependent Reoxidation by NAD+ Catalyzed by Ferredoxin-NAD+ Reductase (Rnf).

Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 µm ferredoxin or 0.42 µm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD(+) reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H(+) = butyryl-CoA + NAD(+) with Km = 1.4 µm ferredoxin or 2.0 µm flavodoxin. This reaction requires Na(+) (Km = 0.12 mm) or Li(+) (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na(+) pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.

Structure and Function of 4-Hydroxyphenylacetate Decarboxylase and Its Cognate Activating Enzyme.

4-Hydroxyphenylacetate decarboxylase (4Hpad) is the prototype of a new class of Fe-S cluster-dependent glycyl radical enzymes (Fe-S GREs) acting on aromatic compounds. The two-enzyme component system comprises a decarboxylase responsible for substrate conversion and a dedicated activating enzyme (4Hpad-AE). The decarboxylase uses a glycyl/thiyl radical dyad to convert 4-hydroxyphenylacetate into p-cresol (4-methylphenol) by a biologically unprecedented Kolbe-type decarboxylation. In addition to the radical dyad prosthetic group, the decarboxylase unit contains two [4Fe-4S] clusters coordinated by an extra small subunit of unknown function. 4Hpad-AE reductively cleaves S-adenosylmethionine (SAM or AdoMet) at a site-differentiated [4Fe-4S]2+/+ cluster (RS cluster) generating a transient 5'-deoxyadenosyl radical that produces a stable glycyl radical in the decarboxylase by the abstraction of a hydrogen atom. 4Hpad-AE binds up to two auxiliary [4Fe-4S] clusters coordinated by a ferredoxin-like insert that is C-terminal to the RS cluster-binding motif. The ferredoxin-like domain with its two auxiliary clusters is not vital for SAM-dependent glycyl radical formation in the decarboxylase, but facilitates a longer lifetime for the radical. This review describes the 4Hpad and cognate AE families and focuses on the recent advances and open questions concerning the structure, function and mechanism of this novel Fe-S-dependent class of GREs.